The study of biological specimens with charged particles remains integral to the advancement of the biological sciences owing to its superior resolution compared to optical techniques. However, such systems work in a vacuum environment so typically the specimens must be chemically altered and dehydrated prior to imaging. This presents an obstacle to obtaining reliable information because faithful imaging depends critically on the sample preparation technique and all dehydration techniques produce artifacts (protein loss, shrinkage etc).
Imaging specimens in a pristine hydrated state (even in vacuum) is possible if the sample is frozen, but care must be taken to avoid the formation of ice crystals which cause disruption of the cellular structure. Typically, techniques currently employed attempt to decrease the temperature of the specimen below the freezing point faster than ice crystals can propagate in the media (a cooling rate of approximately 10,000 degrees Kelvin/sec is required), and thus a crystal-free amorphous or “vitreous” region free of artifacts can be achieved. However, the range of environmental conditions under which vitrification can occur is extremely limited and current cryo-preparation techniques have major shortcomings. For example, in slam freezing, where the sample is forced on a cold metal block (Liquid Nitrogen, LN, temperature or colder), the vitreous region is limited to about 5 microns in depth. This is because of the limited thermal conductivity of the water in the sample. Often the features of interest exist deeper than this shallow vitreous region, and hence such features of interest are subject to extensive crystal damage. Moreover, even a thin layer of water on the sample surface can take up a significant fraction of this vitreous region, providing limited or no information about the sample whatsoever. A much more effective approach is to freeze the samples under extremely high pressures of approximately 2100 bar (i.e., about 30,000 psi), where the freezing temperature of water is depressed and the propagation speed of the crystal growth is significantly reduced due to viscosity changes within the material. High pressure freezing is currently a type of “gold standard” for sample preparation, providing vitreous regions having a depth of up to approximately 200 microns within the sample. Unfortunately, high pressure freezers are undesirably expensive (approximately $250,000 USD), and they require delicate, time consuming sample preparation prior loading into the freezer.
As described in “An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation,” J. Microsc. 165, 255-271 (1992), Hanyu et al. found that a vitreous region within a sample undergoing slam freezing could be extended to 15 microns using a microwave assisted slam freezing technique, in which continuous wave (CW) microwave energy was provided in a manner that disrupted the aggregation of water molecules which into pentamer structures (breaking them into monomers) immediately before the onset of a freezing wave, as indicated in FIG. 1A. Hanyu found that microwave assisted disruption of nucleation sites significantly extended the depth of vitrification, and also changed the character of the ice crystals which formed beyond the vitrified zone. More particularly, as indicated in FIG. 1B, at depths beyond 15 microns, the crystals remained substantially smaller (bounded to less than 50 nm) than in the control-case without microwave disruption (where the crystal size increased without bound beyond 5 microns depth).
Hanyu's apparatus, which is shown in FIGS. 1C and 1D, exposed the sample to microwave radiation as the sample underwent free-fall through a waveguide cavity. The exposure of Hanyu's sample to microwave radiation thus occurred during the time it took the sample to free-fall through the cavity, just prior to impinging on a LN cooled copper block disposed beyond a lower border of the cavity. Unfortunately, Hanyu's apparatus was undesirably limited with respect to the manner in which applied microwave energy interacted with the propagation of a freezing wave within the sample. Furthermore, Hanyu's apparatus was quite cumbersome in terms of its size, configuration, and difficulty of integration with standard microscopy systems or microscopes, and lack of scalability. Additionally, Hanyu's cooling head needed to be re-heated and polished between each sample, so Hanyu's apparatus is not well suited to correlative microscopy.
A need exists for a system, apparatus, device and method that provides greatly improved performance over slam or plunge freezing by way of the selective application of microwave energy to a sample during slam freezing, which provides vitrification depths of up to tens of microns or more, which can be readily integrated with standard types of microscopy equipment (e.g., optical microscopes), which is well-suited for correlative microscopy, and which has a cost that is much lower than current high pressure devices.